We thank Dr. Yule Liu from Tsinghua University for providing the PVX-LIC vector. This work was supported by a start-up fund from the Texas A&#38;M AgriLife Research and the Hatch Project TEX0-1-9675 from USDA National Institute of Food and Agriculture to JS.

1. Grow Potato Plants.
<ol>
	<li>Propagate in vitro potato plants in culture tubes (25 x 150 mm) with Murashige and Skoog (MS) media plus&#160;Gamborg&#8217;s vitamin (MS basal salt mixture, Gamborg&#8217;s vitamin, 30 g/L sucrose, 3.5 g/L agar, pH = 5.7). Place the tubes in the growth room under 20&#8211;22 &#176;C, 16 h light/8 h dark photoperiod, and light intensity 120 &#181;mol/m2&#8729;s1. 
	NOTE: New shoots and roots normally develop in 1&#8211;2 weeks from plants. Propagate plants with fresh MS/Gamborg&#8217;s vitamin media every month.</li>
	<li>Four weeks later, transplant in vitro plants into soil and grow them in a greenhouse under 20&#8211;22 &#176;C, 16 h light/8 h dark photoperiod, and light intensity 120 &#181;mol/m2&#8729;s1. 
	NOTE: The plants with newly developed roots and leaves are suitable for transplanting. Maintain moisture for the freshly transplanted plants for the first 3&#8211;4 days.</li>
</ol>
2. Construct the VbMS vectors.
<ol>
	<li>Design and clone the short tandem TM molecules (STTM, Figure 1)22,53 for the miRNA of interest. 
	NOTE: Acquire miRNA sequences based on experimental data or from the miRbase database54,55,56,57,58,59. The miR166 sequence used in this study has been described previously60.
	<ol>
		<li>Design the TM module by inserting a mismatch sequence, normally 5&#8217;-CTA-3&#8217;, into the reverse complement sequence of miRNA at the site corresponding to the 10th&#8211;11th nucleotides of the miRNA. 
		NOTE: For example, the Stu-miR160 sequence is 5&#8217;-UGCCUGGCUCCCUGUAUGCC-3&#8217;61, where the 10th&#8211;11th nucleotides is in bold. The reverse complement sequence (in deoxynucleotides) is 5&#8217;-GGCATACAGGGAGCCAGGCA-3&#8217; and the mismatch bulge insertion site is shown in bold. The TM molecule sequence (deoxynucleotide) should be 5&#8217;-GGCATACAGG-CTA-GAGCCAGGCA-3&#8217;.</li>
		<li>Design primers for cloning the STTM fragment (Figure 1). Use DNA oligonucleotides with the 48-nt spacer sequence as template for PCR cloning. The forward primer consists of a LIC1 linker (5&#8217;-CgACgACAAgACCgT-3&#8217;), the forward sequence of the above designed for TM molecule, and the partial 5&#8217; sequence of the 48-nt spacer (5&#8217;-GTTGTTGTTGTTATGGT-3&#8217;). The reverse primer consists of a LIC2 linker (5&#8217;-gAggAgAagAgCCgT-3&#8217;), the reverse complement sequence of the TM molecule, and a partial reverse complement to the 3&#8217; sequence of the 48-nt spacer (5&#8217;-ATTCTTCTTCTTTAGACCAT-3&#8217;). 
		NOTE: The 48-nt spacer sequence is 5&#8217;-GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAAT-3&#8217;. For example, for STTM-miR160, the forward primer should be 5&#8217;-CgACgACAAgACCgT-GGCATACAGG-CTA-GAGCCAGGCA-GTTGTTGTTGTTATGGT-3&#8217;; the reverse primer should be 5&#8217;-gAggAgAagAgCCgT-TGCCTGGCTC-TAG-CCTGTATGCC-ATTCTTCTTCTTTAGACCAT-3&#8217; (Figure 1).</li>
		<li>Amplify the STTM fragment in a volume of 50 &#181;L by PCR using the synthesized universal 48-nt spacer as the template and a high fidelity DNA polymerase.
		<ol>
			<li>Set up the PCR reaction by mixing 0.5 &#181;L of each primer (40 &#181;M), 0.5 &#181;L of 48-nt spacer oligo (40 &#181;M), 5 &#181;L of 10x PCR buffer, 1 &#181;L of dNTP mixture (10 mM each), 0.1 &#181;L of high fidelity DNA polymerase (10 U/&#181;L ), and 43 &#181;L of ddH2O to a total volume of 50 &#181;L. Perform standard PCR amplification as follows: 94 &#176;C for 3 min, 32 cycles of 94 &#176;C for 45 s, 60 &#176;C for 45 s and 72 &#176;C for 60 s.</li>
		</ol>
		</li>
		<li>Purify the STTM fragment by ethanol precipitation. Add 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH = 4.0) to the PCR products. Mix vigorously and centrifuge at 14,000 x g for 10 min. Remove the supernatant and rinse the pellet with 1 mL of 70% ethanol. Dry the pellet and dissolve it in 20 &#181;L of ddH2O. 
		NOTE: Use DNA electrophoresis to check the amplification of the STTM PCR products.</li>
		<li>Set up the T4 DNA polymerase reaction on ice by mixing 2.5 &#181;L of purified STTM PCR product, 0.5 &#181;L of 10x T4 DNA polymerase buffer, 0.05 &#181;L of 1 M dithiothreitol (DTT), 0.25 &#181;L of 100 mM dATP, 0.1 &#181;L of T4 DNA polymerase (3 U/&#181;L), and 1.6 &#181;L of ddH2O to a total volume of 5 &#181;L. Incubate the mixture at 37 &#176;C for 15 min, and treat the products at 75 &#176;C for 20 min to inactivate the T4 DNA polymerase.</li>
	</ol>
	</li>
	<li>Prepare the PVX-based VbMS construct.
	<ol>
		<li>Digest 5 &#956;g of PVX-LIC plasmid38 with 2.5 &#181;L of SmaI (20 U/&#181;L) in a volume of 100 &#181;L.</li>
		<li>Add an equal volume of phenol:chloroform:isopropanol (25:24:1, pH = 6.7/8.0) to the digested PVX-LIC products and mix vigorously. Centrifuge at 14,000 x g for 10 min and transfer the supernatant to a new centrifuge tube. Add an equal volume of chloroform:isopropanol (24:1) and vortex vigorously. Centrifuge at 14,000 x g for 10 min. 
		NOTE: The PVX-LIC vector harbors the LIC cassette for cloning. The LIC cassette of PVX-LIC vector contains a ccdB gene and a chloramphenicol-resistant gene and needs to be maintained/propagated in the E. coli strain DB3.1 using LB medium containing kanamycin (50 &#956;g/L) and chloramphenicol (15 &#956;g/L).</li>
		<li>Transfer the supernatant to a new centrifuge tube. Add 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH = 4.0) and mix vigorously. Centrifuge at 14,000 x g for 10 min and remove the supernatant.</li>
		<li>Rinse the pellet with 1 mL of 70% ethanol and vortex vigorously. Centrifuge at 14,000 x g for 10 min and remove the supernatant. Dry the pellet and dissolve the digested PVX-LIC plasmid with 100 &#181;L of ddH2O.</li>
		<li>Set up the T4 DNA polymerase reaction on ice by mixing 2.5 &#181;L of digested PVX-LIC vector DNA, 0.5 &#181;L of 10x T4 DNA polymerase buffer, 0.05 &#181;L of 1 M DTT, 0.25 &#181;L of 100 mM dTTP, and 0.1 &#181;L of T4 DNA polymerase (3 U/&#181;L) in a total volume of 5 &#181;L. Incubate the mixture at 37 &#176;C for 15 min and treat the products at 75&#8201;&#176;C for 20 min to inactivate the T4 DNA polymerase.</li>
	</ol>
	</li>
	<li>Clone the STTM sequence into the PVX-LIC vector using a LIC reaction. Mix the T4 DNA polymerase-treated STTM PCR products (5 &#181;L) and the T4 DNA polymerase-treated PVX-LIC plasmids (5 &#181;L). Incubate at 70 &#176;C for 5&#8201;min, cool down to 22 &#176;C at a ramp of 0.1 &#176;C/s, and keep at 22 &#176;C for 30 min using a PCR machine. 
	NOTE: Extend the incubation time to overnight at 4 &#176;C to achieve higher efficiency of LIC cloning.</li>
	<li>Transform 5 &#181;L of the LIC reaction products into the E. coli DH5&#945; and grow on an LB plate containing 50 &#956;g/mL kanamycin62,63. Pick and verify positive colonies by PCR with the cloning primers and universal primer for the PVX-LIC vector followed by&#160;sequencing.
	<ol>
		<li>Perform colony PCR with the forward primer for PVX-LIC (5&#8217;-GTGTTGGCTTGCAAACTAGAT-3&#8217;) in combination with the reverse primer for STTM cloning to identify positive clones. The size of the PCR band should be ~300 bp. 
		NOTE: Verify the sequences of STTM fragments by terminator cycle sequencing64,65.</li>
	</ol>
	</li>
	<li>Isolate the PVX-STTM plasmids from the validated clones and transform them into Agrobacterium strains GV3101, GV2260, or EHA10562,63. Verify Agrobacterium colonies by PCR. 
	NOTE: Confirm Agrobacterium colonies by PCR using the forward primer for PVX-LIC and the reverse primer for STTM cloning.</li>
</ol>
3. Perform PVX-based VbMS assay in potato plants.
<ol>
	<li>Transplant 4-week-old in vitro potato plants into soil. The transplanted plants will be subjected to VbMS assay 3&#8211;4 days later.</li>
	<li>Inoculate potato plants with Agrobacterium containing the PVX-STTM plasmids. 
	NOTE: For the VbMS assay in potato, Agrobacterium-mediated infiltration and toothpick-scratching inoculation are performed simultaneously.
	<ol>
		<li>Pick and inoculate positive transformants containing PVX-STTM vectors into 50 mL of liquid LB containing 50 &#956;g/mL kanamycin and 50 &#956;g/mL rifampicin. Grow in a 28 &#176;C incubator at 220 rpm for 16 h until OD600 = 1.0.</li>
		<li>At the same time, streak the positive Agrobacterium colonies onto at least two new LB plates containing 50 &#956;g/mL kanamycin and 50 &#956;g/mL rifampicin and grow at 28 &#176;C for 1 day. Include the PVX-LIC38,66 vector as a control and PVX-GFP67,68 to monitor virus spread.</li>
		<li>Collect the Agrobacterium liquid culture by centrifugation at 3,400 x g for 10 min at room temperature. Resuspend Agrobacterium cells with an equal volume of infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 &#181;M acetosyringone, pH = 5.6) and adjust to OD600 = 1.0. Incubate at room temperature for 6 h.</li>
		<li>Infiltrate the Agrobacterium culture into the abaxial side of fully expanded leaves with a 1 mL needleless syringe.
		<ol>
			<li>Flip and hold a leaf with one hand, then use one finger to support the leaf lamina from the adaxial side at the infiltration site. Keep the syringe vertical to the leaf surface with the other hand and infiltrate the Agrobacterium culture into the abaxial side of lamina.</li>
		</ol>
		</li>
		<li>Scrape the Agrobacterium culture from the LB plates and scratch the stem surface of the first one or two internodes of the infiltrated potato plants with a toothpick. Gently scratch the epidermis of the stem. Avoid piercing through the stem, which may cause severe damage to the plants.</li>
	</ol>
	</li>
	<li>Grow the infiltrated plants at 22 &#176;C with a 16 h light/8 h dark photoperiod and light intensity of 120 &#956;mol/m2&#8729; s1 in a greenhouse. 
	NOTE: Phenotypes caused by miRNA silencing usually appear in 2&#8211;4 weeks postinoculation (Figure 2, Figure 3). It typically takes 10&#8211;20 days for the VbMS phenotype to appear after infiltration. The VbMS phenotype depends on the properties of the specific miRNA and target genes, growth conditions, and potato varieties.</li>
</ol>
4. Perform expression analysis.
<ol>
	<li>When phenotypes appear at 2&#8211;4 weeks postinoculation, collect tissues such as shoots, leaves, flowers, or roots with phenotypes from the VbMS plants and tissues from the control plants with scissors. Isolate total RNAs from the collected tissues.</li>
	<li>Check the RNA quality by electrophoresis62,63 and quantify the RNA concentration by measuring the OD260 absorbance with a spectrophotometer.</li>
	<li>Use stem-loop real-time reverse transcription PCR (RT-PCR) to analyze the miRNA expression.
	<ol>
		<li>For specific miRNAs, design a stem-loop reverse transcription primer. Stem-loop RT primers contain a universal 5&#8217; backbone and a 3&#8217; 6-nt extension of a specific miRNA. Design a 5&#8217; universal backbone (5&#8217;- GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGAC-3&#8217;) that forms a stem-loop structure. (The upper case corresponds to an inverse-repeated sequence and the lower case to the loop region).</li>
		<li>Part of the 5&#8217; backbone sequence that forms a loop serves as the reverse primer for subsequent PCR amplification (bold-italic sequence in the backbone sequence). Add a 6-nt extension sequence that is reverse-complementary to the 3&#8217; end of the miRNA of interest to the stem-loop primer to provide specificity for reverse transcription (Supplemental Figure 1A). 
		NOTE: Design the stem-loop reverse transcription primer as described by Chen et al.69 and Erika Varkonyi-Gasic et al.70,71,72. For example, the stem-loop reverse transcription primer for Stu-miR160 is designed as 5&#8217;-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGCATA-3&#8217;. The stem-loop reverse transcription primer for the potato miR165/166 family is designed as 5&#8217;-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGGG(A/G)A-3&#8217;. (The bold uppercase sequences provide the specificity of reverse transcription for specific miRNAs).</li>
		<li>Set up the reverse transcription reaction by mixing 50&#8211;200 ng of total RNA, 1 &#181;L of the stem-loop reverse transcription primer (100 &#956;M), 2 &#181;L of 10x buffer, 0.2 &#181;L of RNase inhibitor (40 U/&#181;L), 0.25 &#181;L of dNTPs (10 mM each), and 1 &#181;L of reverse transcriptase (200 U/&#181;L) on ice. Add nuclease-free ddH2O to a total volume of 20 &#181;L.</li>
		<li>Perform reverse transcription using the pulsed reverse transcription procedure. Incubate the reverse transcription reaction mixture at 16 &#176;C for 30 min, perform a temperature cycle of 30 &#176;C for 30 s, 42 &#176;C for 30 s and 50 &#176;C for 1 s, for a total of 60 cycles, and inactivate the reverse transcriptase by incubation at 85 &#176;C for 5 min.</li>
		<li>For real-time PCR analysis of miRNA expression, design the forward primer based on the miRNA sequence but do not include sequences that overlap with the above designed stem-loop reverse transcription primer. Add a 3&#8211;7-nt extension to the 5&#8217; of the forward primer to optimize the length, melting temperature, and GC content (Supplemental Figure 1). 
		NOTE: The universal reverse primer is 5&#8217;-GTGCAGGGTCCGAGGT-3&#8217;. For example, the stem-loop reverse transcription primer for Stu-miR160 is designed as 5&#8217;-CGGCTGCCTGGCTCC-3&#8217;. The stem-loop reverse transcription primer for miR165/166 family is 5&#8217;-CGGCTCGGACCAGGCTT-3&#8217;. The bold sequences serve as 5&#8217; extensions for primer optimization. The stem-loop reverse transcription primers and the real-time PCR primers for miRNA can be designed using the miRNA Primer Design Tool73.</li>
	</ol>
	</li>
	<li>Synthesize cDNAs of the target mRNAs by standard reverse transcription PCR (RT-PCR). Incubate the reverse transcription reaction mixture at 37 &#176;C for 60 min and inactivate the reverse transcriptase by heating to 85 &#176;C for 5 min. 
	NOTE: (1) Predict target mRNAs using the psRNATarget program74 if the target mRNAs are not known. (2) The universal reverse transcription primer for mRNA is an anchored primer 5&#8217;-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3&#8217;.</li>
	<li>Set up the real-time PCR reaction for both&#160;miRNA of interest and the target mRNAs. Mix 0.5 &#181;L of template cDNA, 5 &#181;L of 2x real-time PCR buffer with SYBR green, 0.05 &#181;L of each forward and reverse primer (40 &#181;M), and 4.4 &#181;L of ddH2O in a total volume of 5 &#181;L on ice.</li>
	<li>Incubate at 95 &#176;C for 3 min, 40 cycles of 95 &#176;C for 3 s and 60 &#176;C for 30 s. Perform a subsequent melting curve analysis as follows: Incubate at 95 &#176;C for 15 s, cool down to 60 &#176;C at a ramp of 20 &#176;C/s, keep at 60 &#176;C for 60 s, heat to 95 &#176;C at a ramp of 0.2 &#176;C/s, and keep at 95 &#176;C for 15 s. Analyze the Ct-values using the &#916;&#916;Ct methods76,77 and plot the means with standard errors. 
	NOTE: (1) The real-time PCR primers for target mRNAs can be designed as described75. (2) Potato polyubiquitin 10 gene can serve as an internal control for normalization in potato. The forward primer for the potato polyubiquitin 10 gene is 5&#8217;-ATGTTGCCTTTCTTATGTGTGGTTG-3&#8217; and the reverse primer 5&#8217;-TTATTTATTCACATAAACGACAGTTCAACC-3&#8217;. (3) For real-time PCR analysis, contamination and primer-dimer formation may generate false positive results. To monitor nonspecific amplification and increase the liability of real-time PCR analysis, it is recommended to include controls without template and reverse transcriptase for real-time PCR assays.</li>
</ol>

We present a PVX-based miRNA silencing system to characterize the function of endogenous miRNAs in potato by integrating the STTM design into the PVX vector. The VbMS system proved to be effective in silencing miRNA165/166 in potato, a highly conserved miRNA family across plant species.
The TM approach has been developed to interfere with the expression of miRNAs based on an artificial miRNA target mimic that is designed to create a mismatch loop at the expected cleavage site within the miRNA ...

Plant microRNAs (miRNAs) are characterized as 20&#8211;24 nucleotide-long, nuclear-encoded regulatory RNAs1 and play fundamental roles in almost every aspect of plant biological processes, including growth and development2,3, photosynthesis and metabolism4,5,6,7, hormone synthesis and signaling8,9, biotic and abiotic responses10,11,12,13, and nutrient and energy regulation14,15. The regulatory roles of plant miRNAs are well-programmed and fulfilled typically at post-transcriptional levels by either cleaving or translationally repressing the target mRNAs.
Tremendous progress has been made towards&#160;identification, transcriptional profiling, and target prediction of miRNAs in potato16,17,18,19,20,21. However, the functional characterization of miRNAs in plants, including potato, has lagged behind other organisms due to the lack of efficient and high-throughput genetic approaches. It is challenging to perform functional analysis of individual miRNA by standard loss-of function analysis, because most miRNAs belong to families with considerable genetic redundancy22. In addition, a single miRNA can control multiple target genes23 and several different miRNAs can modulate the same molecular pathway collaboratively24,25. These properties make it difficult to characterize the function of a specific miRNA or a miRNA family.
Much of the functional analysis of miRNAs has relied heavily on gain-of-function approaches that have obvious limitations. The artificial miRNA (amiRNA) method exploits the endogenous primary transcripts (pri-miRNAs) to produce miRNAs at a high level, leading to inhibition of target gene expression26,27,28,29. However, activation tagging and miRNA overexpression using a strong constitutive 35S promoter often lead to heightened expression of miRNAs that are not representative of in vivo conditions and therefore may not reflect the endogenous function of miRNAs30. An alternative approach has been developed involving expression of miRNA-resistant forms of target genes that contain uncleavable mutations in the binding and/or cleavage sites31,32,33. But this approach can also potentially cause misinterpretation of the phenotype derived from the miRNA-resistant target transgene due to transgenic artifacts. Therefore, conclusions from these gain-of-function studies should be drawn with caution34. Another major limitation of the above-described approaches is that they require transformation, which is labor-intensive and time-consuming. Furthermore, the transgene-dependent approaches are hardly applicable for transform-recalcitrant plant species. Therefore, it is essential to develop a fast and efficient loss-of-function approach to unravel the function&#160;of miRNAs.
To bypass the prerequisite of the transformation procedure, virus-based microRNA silencing (VbMS) has been established by combining the target mimic (TM) strategies with virus-derived vectors. In the VbMS system, artificially designed TM molecules are transiently expressed from a virus backbone, offering a powerful, high-throughput, and time-saving tool to dissect the function of plant endogenous miRNAs35,36. VbMS was initially developed in N. benthamiana and tomato with the tobacco rattle virus (TRV)35,36,37 and has been extended to Arabidopsis, cotton, wheat, and maize using various other virus expression systems, including potato virus X (PVX)38, cotton leaf crumple virus (ClCrV)39, cucumber mosaic virus (CMV)40,41,42, Chinese wheat mosaic virus (CWMV)43, and barley stripe mosaic virus (BSMV)44,45.
Potato (Solanum tuberosum) is the fourth most important food crop and the most widely grown noncereal crop in the world primarily because of its high nutritional value, high energy production, and relatively low input requirements46. Several features of potato make it an attractive dicotyledonous model plant. It is a vegetatively propagated polyploid crop with high outcrossing rate, heterozygosity, and genetic diversity. However, to date, there is no report characterizing the function of miRNAs in potato using VbMS. Here, we present a ligation-independent cloning (LIC)-adapted potato PVX-based VbMS approach to evaluate the function of miRNAs in potato plants38. We selected the miR165/166 family to illustrate the VbMS assay because the miR165/166 family and their target mRNAs and Class III homeodomain/Leu zipper (HD-ZIP III) transcription factors have been extensively characterized22,47,48. The HD-ZIP III genes are key regulators of meristem development and organ polarity, and suppression of miR165/166 function leads to increased expression of the HD-ZIP III genes, resulting in pleotropic developmental defects such as reduced apical dominance and aberrant patterns of leaf polarity22,35,38,41. The readily scorable developmental phenotypes correlated with silencing of miRNA165/166 enable accurate evaluation of the effectiveness of the PVX-based VbMS assay.
In this study, we demonstrate that the PVX-based VbMS system can effectively block the function of miRNAs in potato. Because the PVX-based virus-induced gene silencing (VIGS) system has been established in a number of potato varieties49,50,51,52, this PVX-based VbMS approach can be likely applied to a broad range of diploid and tetraploid potato species.

<table><tbody><tr><td>100 &amp;micro;M dATP and 100 &amp;micro;M dTTP</td><td>Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td><td>TQAC136</td><td></td></tr><tr><td>3 M Sodium acetate, pH 4.0.</td><td>Teknova, Hollister, CA 95023, USA</td><td>#S0297</td><td></td></tr><tr><td>Acetosyringone</td><td>TCI America, Portland, OR 97203, USA</td><td>D2666-25G</td><td></td></tr><tr><td>Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0757-950ML</td><td></td></tr><tr><td>Dimethyl sulfoxide, DMSO</td><td>TCI America, Portland, OR 97203, USA</td><td>D0798-25G</td><td></td></tr><tr><td>DTT</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0281-25G</td><td></td></tr><tr><td>E. coli DB3.1</td><td></td><td></td><td>for maintenance of PVX-LIC and pTRV2e containing the ccdB gene</td></tr><tr><td>E. coli DH5&amp;alpha;</td><td></td><td></td><td>for the destination constructs generated by LIC cloning</td></tr><tr><td>Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed</td><td>ICL Specialty Fertilizers, Summerville, SC 29483, USA</td><td>G99260</td><td></td></tr><tr><td>High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs</td><td>Roche Sequencing and Life Science, Kapa Biosystems,&lt;br/&gt; Wilmington, MA, USA</td><td>7958960001</td><td></td></tr><tr><td>Isoamyl alcohol</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0944-1L</td><td></td></tr><tr><td>Koptec Pure Ethanol &amp;ndash; 200 Proof</td><td>Decon Labs, King of Prussia, PA 19406 , USA</td><td>V1005M</td><td></td></tr><tr><td>MES</td><td>TCI America, Portland, OR 97203, USA</td><td>M0606-250G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ThermoFisher, Waltham, MA 02451, USA</td><td>MFCD00149781</td><td></td></tr><tr><td>M-MuLV Reverse Transcriptase</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>M0253L</td><td></td></tr><tr><td>Nano-drop spectrometer: NanoDrop One&lt;sup&gt;C&lt;/sup&gt; Microvolume UV-Vis Spectrophotometer with Wi-Fi</td><td>ThermoFisher, Waltham, MA 02451, USA</td><td>ND-ONEC-W</td><td></td></tr><tr><td>PCR machine: Bio-Rad MyCycler PCR System</td><td>Bio-Rad, Hercules, California 94547, USA</td><td>170-9703</td><td></td></tr><tr><td>PCR machine: Eppendorf Mastercycler pro</td><td>Eppendorf, Hauppauge, NY 11788, USA</td><td>950030010</td><td></td></tr><tr><td>pH meter</td><td>Sper Scientific, Scottsdale, AZ 85260, USA</td><td>Benchtop pH / mV Meter - 860031</td><td></td></tr><tr><td>Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0.</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0883-400ML</td><td></td></tr><tr><td>Phytagel: Gellan Gum</td><td>Alfa Aesar, Tewksbury, MA 01876, USA</td><td>J63423-A1</td><td></td></tr><tr><td>PVX VIGS vector: PVX-LIC</td><td></td><td></td><td>Zhao et al., 2016</td></tr><tr><td>Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System</td><td>ThermoFisher, Waltham, MA 02451, USA</td><td>4485697</td><td></td></tr><tr><td>Real-time PCR reagent: KAPA SYBR&amp;reg; FAST qPCR Master Mix (2x) Kit</td><td>Roche Sequencing and Life Science, Kapa Biosystems,&lt;br/&gt; Wilmington, MA 01887, USA</td><td>7959389001</td><td></td></tr><tr><td>Restriction enzyme: SmaI</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>R0141S</td><td></td></tr><tr><td>Reverse transcription reagents: qScript cDNA SuperMix</td><td>Quanta BioSciences, Gaithersburg, MD 20877 , USA</td><td>95107-100</td><td></td></tr><tr><td>RNA extraction Kit: E.Z.N.A. Plant RNA Kit</td><td>Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td><td>SKU: D3485-01</td><td></td></tr><tr><td>RNase Inhibitor Murine</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>M0314L</td><td></td></tr><tr><td>RNAzol RT</td><td>Sigma-Aldrich, St. Louis, MO 63103, USA</td><td>R4533</td><td></td></tr><tr><td>Soil: Metro-Mix 360</td><td>Sun Gro Horticulture, Agawam, MA 01001-2907, USA</td><td>Metro-Mix 360</td><td></td></tr><tr><td>T4 DNA polymerase and buffer</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>M0203S</td><td></td></tr></tbody></table>

potato virus x-based microrna silencing (vbms) in potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Figure 2 shows the PVX-STTM165/166 potato plants (Katahdin) with ectopic growth of leaf tissues from the abaxial side of leaf lamina along the veins. More severe phenotypes such as trumpet-shaped leaf formation have also been observed. In contrast, no phenotypic abnormality was observed in the PVX control plants. These results show that the VbMS system was effective in suppressing endogenous miRNA function in tetraploid potato plants and the PVX-VbMS system was a robust genetic tool to deter...

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Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

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Research

JoVE Journal

Environment

3.1K Views.  Texas A&M University System. We presenteren een gedetailleerd protocol voor het op aardappelvirus X (PVX) gebaseerde microRNA-uitschakelingssysteem (VbMS) om endogene microRNA's (miRNA's) in aardappelen functioneel te karakteriseren. Target mimic (TM) moleculen van miRNA van belang worden geïntegreerd in de PVX-vector en tijdelijk uitgedrukt in aardappel om de doel miRNA- of miRNA-familie tot zwijgen te brengen.

Video: Potato Virus X-Based microRNA Silencing VbMS In Potato.

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengineering

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Genetics

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Immunology and Infection

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Biology

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Biochemistry

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

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